Positron emission tomography (PET) is a non-invasive imaging modality that utilizes positron-emitting radionuclides (C-11, N-13, O-15 and F-18). For example, F-18 PET has a number of attributes that make it clinically attractive, including 100% positron efficiency, a very high specific radioactivity, and a short half-life of ˜110 min. However, the short half-life of F-18 and the poor nucleophilicity of fluoride render it difficult to incorporate F-18 in complex molecules. Currently, radiochemistry is a major limiting factor for the field of PET. Despite recent advances, challenges exist for improving F-18 incorporation with respect to reaction rates, efficiency, and selectivity.
The inventors have previously described tetrazine-trans-cyclooctene ligation (‘ITCO ligation’) as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis (M. L. Blackman, M. Royzen and J. M. Fox, J. Am. Chem. Soc. 2008, 130, 13518-13519) trans-Cyclooctene derivatives are readily prepared from cis-cyclooctenes using a photochemical flow-reaction that the inventors developed (M. Royzen, G. P. A. Yap and J. M. Fox, J. Am. Chem. Soc. 2008, 130, 3760-3761). The inventors have found that 3,6-diaryl-s-tetrazines offer an excellent combination of fast reactivity and stability for both the conjugate and starting material. In particular, 3,6-di(2-pyridyl)-s-tetrazines have been shown to display excellent characteristics. Thus, the reaction between trans-cyclooctene and 1a proceeds with a rapid rate (k2˜2000 M−1 s−1 in 9:1 MeOH:water), and is successful in cell media and cell lysate. 3,6-Di(2-pyridyl)-s-tetrazines can easily be functionalized as their amido derivatives (1b), which display excellent stability toward water and biological nucleophiles.

Because of the fast rate of reactivity, the TTCO-ligation offers opportunities for the rapid conjugation of radionuclides to biomolecules, both of which are often available only at low concentration.